Diabetic kidney disease (DKD) has been shown to be associated with an excess risk of cardiovascular death. Inflammation has been considered central to type 2 diabetes (T2D) pathophysiology, and inflammation markers have been linked to cardiovascular disease. The serum and urinary IL-18 levels were significantly elevated in patients with T2D; however, whether interleukin 18 (IL-18) are associated with the severity of arterial stiffness remains to be determined. This study examined the relationship of IL-18 levels with pulse wave velocity (PWV) as a reflector for arterial stiffness in patients with T2D.A total of 180 participants with T2D who had undergone PWV examination were enrolled. Serum and urinary IL-18 levels were measured using sandwich enzyme linked immunosorbent assay (ELISA) kits. Arterial stiffness was determined by carotid–femoral PWV (cf-PWV) and carotid–radial PWV (cr-PWV).r = 0.418, p < 0.001); however, we found no significant correlation between urinary IL-18 and cf-PWV in diabetic subjects without DKD. In addition, we found no significant correlation between urinary IL-18 and cr-PWV in participants with T2D with or without DKD. Moreover, the association remained significant when controlling for arterial stiffness risk factors, urinary albumin-to-creatinine ratio and estimated glomerular filtration rate. cf-PWV was greater in the higher group of urinary IL-18 than in the lower group. Nevertheless, we found no significant correlation between serum IL-18 and cf-PWV in participants with T2D.The urinary IL-18 levels correlated positively with cf-PWV in patients with T2D with DKD (The urinary IL-18 levels appear to be associated with greater cf-PWV, suggesting the link between urinary IL-18 and arterial stiffness in patients with T2D. The association between cardiovascular disease (CVD) and diabetes has been known for decades. Clinical and experimental studies have provided evidence and probable mechanisms that link diabetes to increased atherosclerosis. In particular, patients with T2D had more extensive atherosclerotic CVD (ASCVD) and more non-calcified plaques than patients with T1D. Further experimental studies are needed to elucidate these questions , 4. DespThe relationship of inflammation to insulin resistance is considered central to T2DM pathophysiology , 6. MarkUnlike renal and retinal microvascular disease, there is no pathological fingerprint identifying a distinct atherosclerosis or arterial media calcification in the setting of diabetes. Pulse wave velocity (PWV) is assessed by measuring transit distance and transit time between two sites in the arterial system and taking their ratio. Carotid–femoral PWV (cf-PWV) is the current clinical gold standard measurement of arterial stiffness and has been established as a cardiovascular risk marker .The aim of this study was to examine the relationship of IL-18 levels with PWV as a reflector for arterial stiffness and CVD.The study protocol was approved by Institutional Ethical Committee of Nanjing Medical University (approved No. 2019KY097), and written informed consent was obtained from all subjects. The study conforms to the principles outlined in the Declaration of Helsinki. A total of 180 subjects for this study were enrolled from patients with T2D of the Department of Internal Medicine at the Second Affiliated Hospital of Nanjing Medical University, who had undergone PWV examination between January 2020 and December 2020. Subjects were diagnosed as having T2D according to the WHO criteria and provided multiple morning urine and blood samples for assessment of urinary albumin-to-creatinine ratio (UACR) and estimated glomerular filtration rate (eGFR). On the basis of multiple UACR and eGFR measurements, subjects were classified as T2D without DKD  or T2D with DKD . Subjects with acute inflammatory diseases or malignant neoplasm were excluded, because the levels of inflammation can be markedly enhanced by such disease. Hypertension was defined as a blood pressure (BP) ≥ 130/80 mmHg or current use of antihypertensive medications.Each individual provided blood and morning spot urine samples at baseline for biochemical measurements. Samples were centrifuged at 3,000 rpm at 4°C for 15 min, and aliquots were stored at −80°C if not analyzed immediately. Serum and urinary IL-18 levels were measured using sandwich ELISA kits . Urinary IL-18 was normalized by urinary creatinine (UCr). The intra- and inter-assay coefficient of variations (CVs) for IL-18 were both less than 10%. The sensitivity of the assay was 5.47 pg/ml. These parameters were measured twice for each individual, and geometric mean was used as the baseline value in the analysis.cf-PWV and carotid–radial PWV (cr-PWV) were determined on a fasting state in the morning under room temperature (21°C–25°C) using the Complior Analyzer device  according to the manufacturer’s introduction. Coffee, tea, or nitrates were not allowed within 2 h, and long-acting nitrates were restrained for 12 h before measurement. An experienced technician from the Second Affiliated Hospital of Nanjing Medical University performed the test for all participants. Before measurement, patients rested for 10 min and had their BP measured using a validated oscillometric device . Three probes were placed in a place of palpable pulse of the carotid, femoral, and radial artery, respectively. Ten consecutive recordings were averaged to calculate the transit time using the intersecting tangent algorithm. Carotid–femoral and carotid–radial distances were calculated as direct measurements multiplied by 0.8. Any measurement with a tolerance value more than 3 ms was considered invalid.t-test for normally distributed variables and a Mann–Whitney U-test for non-normally distributed variables. Associations between IL-18 levels and characteristics of type 2 diabetes were examined by Pearson correlation analysis for continuous variables and by Spearman correlation test for categorical variables. Association between urinary IL-18 and cf-PWV was determined by a multivariable linear regression analysis with a stepwise backward method. Mean cf-PWV was compared across the median of the urinary IL-18 levels by the general linear model, followed by covariance analysis. A p-value of less than 0.05 was taken to be statistically significant.All analyses were performed with SPSS 25 software package. Because the distribution of the IL-18 levels appeared to be left-skewed, they were normalized by log-transformation. Comparisons between groups were performed by using an unpaired Student’s 2, HbA1c level of 8.30% , and eGFR of 101.77  ml/min/1.73 m2. A proportion had albuminuria (35.6%) and hypertension (40%).Clinical characteristics in 2, the proportion of male sex, BMI, systolic blood pressure (SBP), diastolic blood pressure (DBP), triglyceride, and serum creatinine were all significantly higher in the group of diabetic subjects with DKD than in the group without DKD. Subjects with DKD had significantly lower levels of albumin and high-density lipoprotein (HDL) compared with subjects without DKD. Interestingly, the urinary levels of IL-18 and cf-PWV were significantly elevated in subjects with DKD compared with those without DKD , whereas the serum IL-18 levels and cr-PWV were not different in the two groups.According to the presence or absence of DKD, as described by UACR ≥ 30 mg/g and/or eGFR < 60 ml/min/1.73 mr = 0.224, p = 0.002), male sex , duration of T2D , SBP , hemoglobin , albumin , eGFR , UACR ≥ 30 mg/g , and serum IL-18  in participants with T2D. However, we found no significant correlation between urinary IL-18 and BMI, DBP, fast blood glucose, HbA1c, total cholesterol, triglyceride, HDL, low-density lipoprotein (LDL), or serum creatinine in participants with T2D. In the meantime, urinary IL-18 correlates with hemoglobin , albumin , eGFR , and serum IL-18  in diabetic subjects with DKD. On the other hand, urinary IL-18 correlates with age , male sex , hemoglobin , and serum creatinine  in diabetic subjects without DKD.By univariate linear regression analysis in r = 0.309, p < 0.001); however, we found no significant correlation between urinary IL-18 and cr-PWV in participants with T2D. Moreover, we only found significant correlation between the urinary IL-18 levels and cf-PWV  in diabetic subjects with DKD. We found no significant correlation between urinary IL-18 and cf-PWV in diabetic subjects without DKD. Nevertheless, we found no significant correlation between serum IL-18 and cf-PWV or cr-PWV in participants with T2D, diabetic subjects with DKD, or diabetic subjects without DKD.We performed univariate analysis of the relationships between the parameters of arterial stiffness and the IL-18 levels in patients with type 2 diabetes Table 2.r = 0.219, p = 0.004), none of such association was significant when urinary IL-18 and the traditional cardiovascular risk factors were simultaneously included in the model (model 3).To further clarify the link between urinary IL-18 and severity of arterial stiffness, we performed multiple regression analysis Table 3,2).Given the association between urinary IL-18 and cf-PWV, the median cf-PWV was compared across the median of the urinary IL-18 levels. cf-PWV was greater in the higher group of urinary IL-18 than in the lower group Table 4.In the present study, we have found in subjects with T2D that the urinary IL-18 levels are associated with increased arterial stiffness as evaluated by cf-PWV. Moreover, the association was independent of the traditional arterial stiffness risk factors and the presence of UACR and eGFR. On the other hand, no significant correlation between serum IL-18 and cf-PWV was found in subjects with T2D. This is the first study that demonstrates the associations between the IL-18 levels and arterial stiffness in patients with T2D, with UACR and eGFR taken into account.In current study, we found that the urinary IL-18 levels were higher in the group of diabetic subjects with DKD than in the group without DKD. This finding is approximately in line with those studies, suggesting that the higher level of urinary IL-18 has been a promise marker of kidney tubule injury , 12, 16.The serum IL-18 levels are associated with albuminuria and atherosclerosis in patients with T2D . SubjectWe have found that the higher IL-18 levels are associated with greater cf-PWV, suggesting link with arterial stiffness. When controlling for age and gender, urinary IL-18 was significantly associated with cf-PWV, and the association was independent of traditional arterial stiffness risk factors . The assUrinary IL-18 is produced within the kidney tissue in response to injury and inflammation . UrinaryThere are some limitations for the current study. First, we cannot currently determine the causal relationships between the IL-18 levels and greater arterial stiffness because of the cross-sectional design. Second, this study included substantial number of patients with T2D on medications, requiring studies to separate the effects of such medications. Furthermore, the source of urinary IL-18 in patients with T2D is not known. The elevated IL-18 levels in diabetic kidney tissue or infiltrated macrophage in the kidney may be responsible. Further experimental studies are needed to elucidate these questions.In conclusion, we have demonstrated an association between the higher urinary IL-18 level and greater cf-PWV, suggesting the link between IL-18 and arterial stiffness in patients with T2D. This finding might offer a clue to understand the role of IL-18 in the development of CVDs.The original contributions presented in the study are included in the article/supplementary material. Further inquiries can be directed to the corresponding authors.The studies involving human participants were reviewed and approved by Institutional ethical committee of Nanjing Medical University. The patients/participants provided their written informed consent to participate in this study.Concept the study: JY and YZ; data acquisition: CS, AH, XW, and XZ; data analysis/interpretation: CS, LW, LJ, and YZ; statistical analyses: LJ, JY, and YZ; drafting the work and revising: JY and YZ. All authors contributed to the article and approved the submitted version.Natural Science Foundation of Jiangsu province: General program BK20201497 to YZ, and National Natural Science Foundation of China: General program 81873618 to JY.The authors thank the investigators, staff, and trial participants for dedication and commitment to the trial.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Helicobacter pylori infection and aspirin-induced ulcers in rat’s stomachs was performed using docosahexaenoic acid (DHA)-loaded chitosan/alginate (CA) nanoparticles (NPs) developed by ionotropic gelation method. The physicochemical analyses of the composite NPs were performed by scanning electron microscopy, Fourier transform infrared spectroscopy, zeta potential, X-ray diffraction, and atomic force microscopy. The encapsulation efficiency of AMX was increased to 76% by incorporating DHA, which resulted in a reduction in the particle size. The formed CA-DHA-AMX NPs effectively adhered to the bacteria and rat gastric mucosa. Their antibacterial properties were more potent than those of the single AMX and CA-DHA NPs as demonstrated by the in vivo assay. The composite NPs attained higher mucoadhesive potential during food intake than during fasting (p = 0.029). At 10 and 20 mg/kg AMX, the CA-AMX-DHA showed more potent activities against H. pylori than the CA-AMX, CA-DHA, and single AMX. The in vivo study showed that the effective dose of AMX was lower when DHA was included, indicating better drug delivery and stability of the encapsulated AMX. Both mucosal thickening and ulcer index were significantly higher in the groups receiving CA-DHA-AMX than in the groups receiving CA-AMX and single AMX. The presence of DHA declines the pro-inflammatory cytokines including IL-1β, IL-6, and IL-17A. The synergistic effects of AMX and the CA-DHA formulation increased the biocidal activities against H. pylori infection and improved ulcer healing properties.Encapsulation of amoxicillin (AMX) for drug delivery against  Helicobacter pylori, a Gram-negative bacterium with carcinogenic potential, is a major cause of gastric malignancies such as MALT lymphoma and gastric adenocarcinoma (H. pylori eradication could reduce the risk of metachronous gastric cancer by 50% (H. pylori infection is clarithromycin (CLA) and amoxicillin (AMX) or metronidazole (MET) in combination with a proton pump inhibitor (PPI)  . Howeveror (PPI) . As the failures .H. pylori showed resistance to MET and CLA and a less degree to AMX  composite NPs were used to entrap AMX and DHA. The physicochemical characterizations of the NPs were assessed. The antibacterial activity was determined using an in vitro growth inhibition assay, followed by the evaluation of the mucoadhesive potential of the FITC-labeled NPs. In the in vivo experiments, an aspirin-induced gastric ulcer was induced in rats and the rats were infected with H. pylori. To determine the biocidal effects against H. pylori and the curing effect of the composite NPs on the induced ulcer, the H. pylori colonization, ulcer area, and histopathological changes were monitored.Fatty acids (FAs) that exhibit antibacterial activity against multidrug-resistant (MDR) bacteria could provide the next generation of antibacterial agents for the treatment and prevention of bacterial infections . A combill death . To datecarriers . ChitosaIn the A CA-based NP was prepared based on a previous study by in vivo study was conducted to test CA-DHA, CA-AMX, and CA-DHA-AMX NPs with the following concentrations of the components: CA (1.0% v/v), DHA (2.0% v/v), and AMX at two concentrations (10 and 20 mg/kg).CA-DHA NPs were prepared in several formulations including chitosan , DHA , and AMX . The The physicochemical properties of the composite NPs were evaluated using a scanning electron microscope (SEM), Fourier transforms infrared spectroscopy (FTIR), X-ray diffraction (XRD), atomic force microscopy (AFM), zeta potential, and swelling index (SI) analysis, and the methods are described in detail in The content of DHA and AMX in the recovered solution was measured using a UV/Vis spectrophotometer (JENWAY/6105) at 205 and 272 nm, respectively. To compare the loading of AMX with the hydrophobic and hydrophilic compounds, it was tested in the emulsion and aqueous environments. A detailed explanation of the method used to determine the drug content can be found at H. pylori (H.12.5) in vitro. Briefly, 10 μl of the bacterial suspension (109 CFU/ml) was added to Columbia broth medium (190 μl) and the mixture was incubated under microaerophilic conditions for 6, 12, and 24 h. The growth inhibition assay was performed in vitro. Growth inhibition was estimated from the absorbance of the medium at a wavelength of 600 nm. in vitro antibacterial assay, and the formula for growth inhibition. To evaluate antibacterial activity in vivo, plate colony counts (CFU/gr stomach) and the number of bacteria in the biopsy samples were used  was used to calculate the fluorescence intensity of the adherent FITC-labeled NPs.The in vitro mucoadhesive study. In vivo mucoadhesive activity was performed for two main feeding conditions: fasting and fed. Each of the two groups consisted of four different sub-groups . A total of 24 rats were used to determine the mucoadhesive potential in vivo.One hundred forty-seven male Sprague Dawley rats were involved in this study. Six rats were lost during infection and ulcer induction. Eighteen stomachs from rats were used to evaluate the H. pylori  at 24, 48, and 72 h after ASA ulceration. Infection was confirmed after 2 weeks, and treatment was initiated. Three rats were used to confirm H. pylori colonization and three rats were used for the gastric ulcer confirmation. The in vivo antibacterial activity of different formulations was assessed in thirty rats divided into ten groups at day 7 post-infection  acetylsalicylic acid (ASA) on day 0 to induce gastric ulcers. Bacterial infection was induced by oral administration of nfection .in vivo studies were performed to investigate the ulcer healing activity by macroscopic and microscopic analysis of gastric ulcers by ulcer thickness, ulcer area, ulcer index, and H. pylori eradication was also determined by the direct bacterial counting method. Collagen accumulation and concentrations of inflammatory cytokines, including IL-1β, IL-6, and IL-17A, were determined in the treated and untreated groups on day 14 post-infection. Forty-two rats were randomly divided into seven groups (six rats/group); group 1 , groups 2 and 3: AMX (powder 10 and 20 mg/kg WB), groups 4 and 5: CA-AMX (10 and 20 mg/kg WB), and groups 6 and 7: CA-DHA-AMX (10 and 20 mg/kg WB) to evaluate ulcer healing and H. pylori eradication on day 14. Eighteen rats were divided into six treated groups to evaluate the relapse of infection on day 21 post-infection (three rats/group). One uninfected group was defined as control (n = 3).The 2 were measured at a magnification of 40× and the information was processed using IimageJ software. The area of the gastric ulcer in each section (5 sections/sample) was determined. Gastric histology was evaluated by 1 pathologist who was blinded to the other assays and results.We performed a histopathological examination on gastric biopsies. The ulcer evaluation was done on the whole stomach on day 14 to assess the healing effect. Ulcer indexing was performed according to the previous study by All animal experiments were performed according to accordance with the U.K. Animals (Scientific Procedures) Act, and protocol approved by the Ethics Committee of Ilam Medical University .A whole blood sample was taken from the rats. The presence of IL-1β, IL-6, and IL-17A in serum was measured using ELISA kits according to the manufacturer’s instructions. The intensity was determined at 450 nm using a microplate reader. To ensure consistency of the assay, all plates contained positive control (FBS) and negative control (PBS) samples.Masson’s trichrome staining, which stains collagen fibers blue and faint green, was used to assess collagen accumulation. Naturally, collagen fibers are usually accumulated in the gastric mucosa and submucosa. Immunohistochemistry was performed using antibodies against type I collagen. Sections were incubated with 0.3% hydrogen peroxide in PBS for 30 min and then with 10% normal donkey or goat serum in 0.05 M PBS for 30 min. They were then incubated with polyclonal antibodies labeling collagen I. Each sample was imaged using a Nikon TE 2000 fluorescence microscope . The area of collagen in each section was measured using ImageJ software.t-test with a confidence level of 95% (p < 0.05). Statistical analysis was performed using the Wilcoxon Sum Rank test for the ulcer index. Differences between means were tested for more than two groups with a one-way analysis of variance (ANOVA) followed by a Bonferroni’s post-hoc test. Differences between groups and time points were analyzed with a two-way analysis of variance (ANOVA) and subsequent multiple comparisons using the Bonferroni correction. *p < 0.05; **p < 0.001; ***p = 0.0002; and ****p < 0.0001 indicated statistically significant results.The obtained results were statistically analyzed using Mann–Whitney’s H. pylori eradication properties. Based on the in vitro results, we tested the physicochemical properties of CA-DHA-AMX at the defined concentrations of 1% v/v, 2% v/v, and 60 mg/ml, respectively.This study showed that the NPs were formed by the interactions between the positively charged amino groups of the polycationic agent (chitosan) and the negatively charged polyanionic agent . Based on the strong mucoadhesive properties of CA NPs , we deveAs demonstrated by SEM and FAM, the composite nanoparticles with DHA produced smaller NPs (350 ± 110 nm) with a smoother surface than the DHA-free NPs (600 ± 92 nm) in a concentration-dependent manner. Their spherical shape is due to a hydrophobic group (DHA) in their structure. NPs with smaller sizes were obtained during the oil-in-water micelle structure, resulting in a homogeneous dispersion of DHA .−1, while the peaks of the amino group at 1596 cm−1 and carboxyl groups at 1619 cm−1 disappeared. An ionic interaction was confirmed between the carboxyl group of the alginates and the amino group of chitosan. The new major peak at 1750 cm−1 showed a significant increase in absorption confirming the presence of DHA in CA-based scaffolds. AMX major peaks were observed at 3,440 cm−1 (amide NH and phenol OH stretch), 3,020 cm−1 (benzene ring C-H stretch), 1,770 cm−1 (beta-lactam C-O stretch), 1,680 cm−1 (amide I C-O stretch), 1,500 cm−1 (benzene ring C-C stretch), and 1,480 cm−1 (N-H bend C-N stretch combination band). Characteristic peaks of AMX were also present in the FTIR spectrum of the composite beads with some broadening and reduction in intensity, indicating the absence of the chemical interactions between the drug, polymer, and counter ions after the formation of beads.FTIR spectra of the composite NP CA-DHA-AMX and their components are shown in As shown in AFM analysis revealed that the synthesized NPs were almost monodisperse, without agglomeration ,E. EvideIn a composite polymer, swelling occurs due to water absorption, causing the NPs to expand during water penetration, which leads to an increased crosslinking of the composite polymers . DHA conAn influential factor that affects the effectiveness of drug encapsulation is the effectiveness of drug entrapment. The drug entrapment efficacy of DHA and AMX was also evaluated in the same way as shown in H. pylori and inhibited its colonization in animal models  . Therefore, we examined the composite NPs with 2% v/v DHA. CA-DHA-AMX proved to be more effective against bacteria when the dose of AMX was increased (p = 0.0042), the presence of DHA in the CA-DHA-AMX formulation significantly increased antibacterial activity (p = 0.0009)  . Accorditrations . We alsoncreased . Thus, wncreased . Despite 0.0009) .p = 0.029 and > 0.001 for the release of AMX and DHA, respectively)  .in vivo. This suggested that the antibacterial properties of NPs may be derived from their chitosan coating (p = 0.04)  of chitosan and the polyanionic acid of the bacterial cell wall, leading to membrane disintegration, osmotic pressure disturbance, and eventually cell death  . The lowll death .H. pylori by comparing the antibacterial activities of AMX, CA-AMX, and CA-DHA-AMX. In contrast to the free AMX, the encapsulated AMX showed a significant enhancement of antibacterial activity. Based on the results of this study, there was an additive effect of DHA in CA-DHA-AMX on the antibacterial activity of 10 mg/kg AMX . The antibacterial activity of CA-DHA-AMX was almost the same at the concentrations tested. These results are interesting, and it could be hypothesized that the incorporation of DHA in CA NPs could reduce the effective dose of AMX.The results showed that the most potent antibacterial activity in CA-DHA-AMX was at 20 mg/kg AMX . Statist2 groups of the protonated chitosan under acidic conditions, which are reduced by DHA   . The muc= 0.014) . The pos= 0.014) . At pH =p = 0.029)  and the fed condition (2 h after feeding) is different: 2.5 and 3.0–4.5, respectively. The results showed that the mucoadhesive potential was significantly higher in the fed condition than in the fasting condition . The muc= 0.029) ,C. In co= 0.029) . ChitosaH. pylori. On day 21, H. pylori were found to be completely eradicated with CA-DHA-AMX (10 and 20 mg/kg). We found that DHA significantly increased the efficacy of AMX in the encapsulated formulations. AMX (10 mg/kg) in the encapsulated form significantly increased eradication rates compared with AMX powder 20 mg/kg (p = 0.0184)  . On the  0.0184) .p = 0.009). In this study, DHA was found to accelerate the healing of ulcers induced by ASA.H. pylori infection    . The encctively) . Accordictively) . The patctively) . CA-DHA-ctively) ,F. The sThe macroscopic and microscopic evaluation of each group showed that inflammation decreased in the presence of DHA in different formulations ,D. The iPro-inflammatory cytokines such as IL -6, IL -1β, and IL -17A were significantly lower in the formulations containing DHA than in those without. ELISA results showed that the presence of DHA decreased IL -1β in different formulations. In the CA-DHA-AMX (10 mg/kg) group, the concentration of IL -1β was significantly higher than in the formulations that did not contain it. The CA-DHA-AMX groups were compared with the AMX powder and CA-AMX groups, and the results indicated that DHA was responsible for the anti-inflammatory effect of the composite NPs . The hisH. pylori, with a reduced effective dose of the antibiotic. As a mucoadhesive carrier, chitosan-based nanoparticles are an effective way to deliver acid-sensitive antibiotics such as AMX. The encapsulation of AMX significantly increased its antibacterial activity compared to single AMX, and incorporation of DHA decreased the effective dose. The DHA also decreased the effective dose of AMX in the encapsulated form by increasing its entrapment, which may be due to the modification of bacterial cell walls and its antibacterial activity. The incorporation of DHA into CA-AMX composite NPs enhanced the antibacterial activity in vivo and accelerated the healing of gastric ulcers, which could be attributed to the DHA -mediated dissolution of bacterial cell membrane, macrophage-dependent clearance, and anti-inflammatory effects of DHA. The in vivo anti-H. pylori effects of DHA may also be due to its immunomodulatory activities that elicit biocidal effects on H. pylori.The development of drugs containing acid-sensitive antibiotics such as AMX is difficult with conventional gastric retention formulation techniques. The development of a therapeutically effective gastroprotective formulation of AMX, which has both excellent buoyancy and a suitable release pattern, could allow the targeting of drugs to specific sites in the stomach. Our developed system had none of the disadvantages of a single-dose formulation but offered the advantage of the ease of preparation and sustained release of the drug over an extended period. A simple method of encapsulating AMX in DHA-loaded CA NPs can be used for biocidal effects against The original contributions presented in the study are included in the article/The animal study was reviewed and approved by all animal experiments were performed according to a protocol approved by the Ethics Committee of Ilam Medical University . Written informed consent was obtained from the owners for the participation of their animals in this study.MH: conceptualization, visualization, and supervision. MH and MA: methodology and validation. SK and VK: investigation and data curation. SK, NS and MA: writing – original draft preparation. ME-S and BN: writing – reviewing and editing. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Dental pulp is essential for the development and long-term preservation of teeth. Dental trauma and caries often lead to pulp inflammation. Vital pulp therapy using dental pulp-capping materials is an approach to preserving the vitality of injured dental pulp. Most pulp-capping materials used in clinics have good biocompatibility to promote mineralization, but their anti-inflammatory effect is weak. Therefore, the failure rate will increase when dental pulp inflammation is severe. The present study developed an amorphous calcium phosphate/poly (L-lactic acid)-poly (lactic-co-glycolic acid) membrane compounded with aspirin (hereafter known as ASP/PLGA-ASP/ACP/PLLA-PLGA). The composite membrane, used as a pulp-capping material, effectively achieved the rapid release of high concentrations of the anti-inflammatory drug aspirin during the early stages as well as the long-term release of low concentrations of aspirin and calcium/phosphorus ions during the later stages, which could repair inflamed dental pulp and promote mineralization. Meanwhile, the composite membrane promoted the proliferation of inflamed dental pulp stem cells, downregulated the expression of inflammatory markers, upregulated the expression of mineralization-related markers, and induced the formation of stronger reparative dentin in the rat pulpitis model. These findings indicate that this material may be suitable for use as a pulp-capping material in clinical applications. The preservation of vital dental pulp is essential for maintaining tooth sensation, nutrition, immunity, and dentin formation; these aspects contribute to long-term prognoses ,2. ThereIdeal pulp-capping material for early-stage pulpitis should rapidly inhibit inflammation to create a beneficial microenvironment for dental pulp repair, after which it should promote long-term mineralization to form a hard tissue barrier ,9,10. InThe dental pulp-capping material was designed as a three-layer composite membrane. The lower layer was an ASP/PLGA membrane that contained high concentrations of ASP; the middle layer was an ASP/ACP/PLLA membrane that contained low concentrations of ASP and ACP, and the outer layer was a PLGA membrane. The lower ASP/PLGA membrane can rapidly release aspirin to control pulp inflammation; the middle ASP/ACP/PLLA membrane can perform the long-term release of calcium/phosphorus ions and aspirin to promote the formation of reparative dentin; and the outer PLGA membrane layer improves the superficial hydrophilicity of the composite membrane, which is beneficial to improving the sealing property of the pulp-capping material.Pulpitis is inflammation caused by bacteria or toxins invading the dental pulp in the center of the tooth. Gram-negative bacteria are common microorganisms that cause pulp inflammation and necrosis. Lipopolysaccharide (LPS) is the main molecular component of these bacterial cell walls . In vivow/v) with aspirin at 0.12 wt% PLLA and ACP powders at 10 wt% PLLA were prepared in a 3:1 DCM: DMF solvent mixture. The solution was stirred for 1 h to obtain a uniform suspension, after which this suspension was loaded into a 20 mL syringe and electrospun at 20 kV under a steady flow rate of 1.2 mL/h. The distance between the collection foil and the syringe needle was set as 10 cm. After electrospinning for 2.5 h, the resulting ASP/ACP/PLLA membrane was separated from the collection foil and dried in a desiccator overnight. The dried ASP/ACP/PLLA membrane was cut into small pieces of equal weight and put on the collection foil for further electrospinning.The membrane was fabricated by using a multilayering electrospinning method. Briefly, a PLLA polymer solution . The diameter distribution of the nanofibers was analyzed using the image analysis program ImageJ. An energy-dispersive spectrometer (EDS) was used to determine the chemical composition of the electrospun membrane. The contact angle of the ASP/ACP/PLLA and ASP/PLGA-ASP/ACP/PLLA-PLGA membranes was measured by a contact angle measurement system  with a water droplet size of 0.5 μL. Moreover, transmission electron microscope (TEM) and X-ray diffraction (XRD) were employed here to identify that the ACP we loaded is in amorphous phases. For the TEM  observation, the samples were ultrasonicated in alcohol for 30 min and picked up with copper net films to observe their surface morphologies. Meanwhile, the XRD patterns were obtained from a Rigaku D/Max X-ray diffractometer  with Cu Kα radiation at 40 kV, 120 mA, and a scanning rate of 10°/min from 3° to 70°.In vitro degradation experiments were conducted by monitoring the media pH and mass loss of the electrospun membrane during the course of 21 days. Briefly, 20 mg of the ASP/PLGA-ASP/ACP/PLLA-PLGA membrane were immersed in 3 mL of phosphate buffered solution  and kept at 37 °C incubator shaker. The PBS media were changed every three days with fresh PBS. The replaced PBS was collected, and the pH value of this degradation media was measured by using a pH meter . The mass loss of the composite membrane was examined on 3, 7, 14 and 21 days using an electronic balance, and the membrane was lyophilized for 24 h before measuring.2+, PO43−) and aspirin release from the membrane over a period of 21 days. Before the release experiments, an acceleration study was firstly carried out according to our previous work to calculate the aspirin loading efficiency in the membrane. In order to study the release in vitro, 20 mg of the ASP/PLGA-ASP/ACP/PLLA-PLGA membrane were placed in test tubes with 3 mL of physiological saline  or PBS (for aspirin measurement) and kept in an incubator shaker that is maintained at 37 °C. At predetermined time intervals, the release media in test tubes were collected and replaced with an equal amount of fresh media. The Ca2+, PO43−, and aspirin concentrations were tested by a calcium colorimetric assay kit  and a phosphate ion colorimetric assay kit , as well as a multimode microplate reader  at 278 nm/298 nm, respectively. In vitro release experiments were carried out to analyze the ion release  were provided by the Oral Stem Cell Bank . The DPCs were cultured in an α-modified minimum essential medium  with 10% fetal bovine serum  and 100 U/mL penicillin-streptomycin  at 37 °C in a 5% COThe effect of the composite membrane on DPCs proliferation was assessed by using a Cell Counting Kit-8 (CCK-8) assay . DPCs were seeded at a density of 3 × 103 cells/well in 96-well plates (n = 3 wells per group), cultured, and treated with the culture medium containing ASP/PLGA-ASP/ACP/PLLA-PLGA composite membrane extract, while the α-MEM was used for the control group. At 1, 3, 5, and 7 days, 10 µL of the CCK-8 solution was added to each well. The absorbance was measured at 450 nm by using an Absorbance Microplate Reader . The DPCs were treated with 1 μg/mL of lipopolysaccharide (LPS) to establish a model of inflamed dental pulp stem cells (iDPCs). The cells were treated separately with ASP, ACP/PLLA-PLGA scaffold extract, PLGA-PLLA-PLGA scaffold extract, or ASP/PLGA-ASP/ACP/PLLA-PLGA composite membrane extract; the α-MEM was used for the control group. At 1, 3, 5, and 7 days, CCK-8 assays were conducted as described previously.2 in a humidified incubator for 12 h, photographs were collected by using the inverted microscope at the same observation point to record the distance of cell migration; the distance was measured and analyzed by using ImageJ software.Cell adhesion was observed by using phalloidin/4,6-diamino-2-phenylindole  staining. DPCs were seeded at a density of 1 × 104 cells/well in a 24-well plate with a composite membrane or a glass slide at the bottom and cultured in the α-MEM. After 24 h, the cells were washed and fixed with paraformaldehyde. Phalloidin staining was performed for 30 min, and the nuclei were stained with DAPI for 10 min. After the cells had been washed with phosphate-buffered saline, cell skeletons were observed by using a confocal microscope . The cell scratch test was used to examine the impact of each membrane treatment on DPCs migration. DPCs were seeded at 1 × 105 cells/well in a 6-well plate. When the cells reached 90% confluence, a clear and straight scratch was drawn by using a 1 mL pipette tip on the bottom of the plate; the scratch was measured by using a ruler. Exfoliated cells were washed away using phosphate-buffered saline, and the remaining DPCs were treated with the membrane extract liquid and α-MEM. The initial position and width of the scratch were recorded by photography using an inverted microscope. After culturing at 37 °C with 5% COARS was performed to evaluate intracellular mineral deposition. DPCs were seeded at 6 × 104 cells/well in a 12-well plate and were then treated with LPS. Subsequently, they were cultured with ASP, ACP/PLLA-PLGA membrane extract, or ASP/PLGA-ASP/ACP/PLLA-PLGA composite membrane extract, α-MEM only was used for the control group. When the cells reached approximately 80% confluence, the medium was replaced with osteogenic differentiation media that contained 100 nM dexamethasone, 10 mM sodium β-glycerophosphate, and 10 nM L-ascorbic acid . After being cultured for 14 days, the cells were then fixed in 4% paraformaldehyde, washed with phosphate-buffered saline to remove fixative residues, and stained using a 2% ARS solution . The samples were subjected to a microscopy analysis for the qualitative evaluation of the differentiation. For the quantitative evaluation, ARS was dissolved in 5% perchloric acid; cellular absorbance was then measured at 490 nm using a Varioskan Flash  microplate spectrophotometer.DPCs were seeded at 1 × 105 cells/well in a 6-well plate and were then treated with LPS. Cells were cultured for 7 days in osteogenic media with the membrane extract. The relative expression levels of osteogenic/odontogenic markers alkaline phosphatase (ALP), dentin sialophosphoprotein (DSPP), dentin matrix protein1 (DMP1), Bone Sialoprotein (BSP) and inflammatory cytokines tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), interleukin-6 (IL-6), interleukin-8 (IL-8) were evaluated by using qPCR. Ribonucleic acid (RNA) was extracted from each group of DPCs by using TRIzol (Invitrogen); the total RNA was then reverse-transcribed to cDNA by using the Moloney murine leukemia virus reverse transcriptase . qPCR was performed by using SYBR Green Master Mix  with 0.5 μL cDNA and 200 nM specific primers . The absorbance was measured at 450 nm by using a microplate reader. The concentrations of the cytokines in the cells were analyzed by using Prism 6.0 software.DPCs were cultured for 14 days in osteogenic media with the membrane extract for Western blot assays. The cells were harvested with a protein lysis buffer . The protein concentration was determined by a BCA protein analysis kit . Equal aliquots of 40 µg of total protein were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS–PAGE) and transferred to polyvinylidene difluoride membranes (PVDF) . After blocking in 5% nonfat dry milk for 1 h, the proteins of interest were probed with primary antibodies overnight at 4 °C: DSPP, DMP1, and osteocalcin (OCN) . After being incubated with secondary antibodies for 1 h, the immunoblots were detected by the Western enhanced chemiluminescence blotting kit .2 inhalation. The specimens were then fixed, decalcified, and sectioned (4 μm). Hematoxylin–eosin staining was performed, and DMP1 expression was detected via immunohistochemistry.Healthy upper first molars from Sprague Dawley rats were used to establish the experimental pulpitis model for this investigation. Rats were anesthetized by using intraperitoneal injections of 10% chloral hydrate . Cavity preparation and pulp chamber opening were performed by using high-speed turbine tooth drill burs  with a terminal diameter of 0.5 mm. Coronal pulpal tissue was damaged by using sterile #40 K-files, treated for 30 min with sterile cotton balls that had been soaked in 1 μg/mL of LPS, and then rinse with saline; bleeding was controlled with a small cotton pellet with gentle pressure. The ASP/PLGA-ASP/ACP/PLLA-PLGA composite membrane was placed in the cavity over the exposed pulp. Mineral trioxide aggregate  was used to treat the contralateral cavity as a control. The cavity was then sealed with glass-ionomer cement . After 8 weeks in a specific pathogen-free laboratory, the rats were sacrificed by COp-values < 0.05 were considered statistically significant. Statistical analyses were performed by using SPSS Statistics 23.0 software . The groups were compared by using a one-way analysis of variance. 2+ and PO43−. Compared with aspirin, the ion release rates were somewhat faster, with the observation of an obvious initial burst release. Approximately 75% of the Ca2+ and PO43− were released from the membrane on the first day. After the burst release, the Ca2+ and PO43− were released in a slower but constant pattern until day 21.The in vitro degradation experiment was conducted by measuring the mass loss and media pH variation over a period of 21 days. The mass loss of the electrospun membrane stayed at a slow but constant speed during the whole in vitro experiment. It can be seen from The CCK-8 analysis showed that the DPCs treated with the composite membrane extract grew stably, with proliferation rates higher than the control group on day seven A. DPCs pThe mineralization-inducing ability of the composite membrane was assessed by using ARS. After 21 days of culturing in osteogenic media, mineralized nodules were stained red with ARS. Mineralized nodule formation significantly decreased after LPS treatment, while it increased after the addition of ASP and PLGA-ACP/PLLA-PLGA. The ASP/PLGA-ASP/ACP/PLLA-PLGA group demonstrated significantly more mineralization according to the ARS findings A. The odIn order to assess the anti-inflammatory effects of the composite membrane, the levels of inflammatory biomarkers were measured by using qPCR. The mRNA expression levels of the inflammatory cytokines IL-1β, IL-6, TNF-α, and IL-8 were higher in iDPCs than in DPCs. After treatment with ASP or the ASP/PLGA-ASP/ACP/PLLA-PLGA composite membrane, the mRNA expression levels of inflammatory cytokines significantly decreased in iDPCs A. EnzymeThe in vivo effects of the ASP/PLGA-ASP/ACP/PLLA-PLGA composite membrane as a pulp-capping material was investigated in a rat dental pulpitis model. Pulp tissues treated with LPS became necrotic at eight weeks B,C. NewlDental caries and trauma often lead to dental pulp exposure. The dysplasia of enamel and dentin caused by some genetic diseases may also lead to dental pulp tissue being vulnerable to damage ,22,23. VThe anti-inflammatory drug, ASP, used in this study exerts favorable anti-inflammatory effects by inhibiting cell cyclooxygenase, blocking the conversion of prostaglandins into arachidonic acid, and reducing the production of inflammatory mediators. It can also inhibit the expression of nuclear-factor-κB-mediated inflammatory cytokines , which has important anti-inflammatory effects ,37,38,39Ideally, pulp capping should be able to repair the damaged pulp and the material can be replaced by reparative dentin. Therefore, the pulp-capping material should be biodegradable. Meanwhile, some physical and chemical properties of pulp-capping materials, such as the pH value and calcium ion release, are also crucial because they will have a direct impact on the repair process of dental pulp tissue ,53. ACP In this study, the effect of the composite membrane on iDPCs proliferation and odontoblastic differentiation after LPS treatment was more than the effects of ASP or ACP/PLLA/PLGA scaffolds alone. This finding is potentially the case because the early release of ASP controlled inflammation and created a suitable environment for cell proliferation and differentiation, while the subsequent controlled release of ASP and ACP promoted a microenvironment that aided in pulp tissue repair.The purpose of this paper was to solve the problem of preserving vital pulp in clinical practice. The rat pulp-capping model was used for eight weeks of observations. In future studies, the observation time should be extended, and large animals should be used to further observe the pulp-capping effect of the material. In addition, for the release of anti-inflammatory drugs, such as aspirin, in pulp-capping materials, inflammatory responsive controlled release materials can be further designed for different inflammatory states so as to more accurately regulate the pulp inflammation state. This paper provides a new idea and theoretical basis for the design and application of vital dental pulp preservation materials.In this study, the composite application of aspirin, amorphous calcium phosphate, and PLLA/PLGA scaffold materials was used to develop a pulp-capping material with multiple effects for the repair of the injured dental pulp. The ASP/PLGA-ASP/ACP/PLLA-PLGA composite membrane developed in this study allowed for the sequential release of the anti-inflammatory drug aspirin as well as the calcium/phosphorus ions required for mineralization; The mass loss of the membrane stayed at a slow but constant speed, with a relatively stable pH value during degradation; it also promoted the proliferation and odontogenic differentiation of iDPCs, while inhibiting inflammatory cytokine release. It had the dual effects of reducing inflammation and promoting mineralized barrier formation. The ASP/PLGA-ASP/ACP/PLLA-PLGA composite membrane represents a promising vital pulp conservation material with the potential for clinical applications.The application of composite materials for pulp therapy can combine the advantages of multiple materials, but it also faces great challenges because the mechanism of action between composites is complex and needs to be further explored. We will further evaluate the biological properties of the material and conduct long-term in vivo studies to assess the effectiveness of ASP/PLGA-ASP/ACP/PLLA-PLGA composite membrane as a vital pulp-capping agent in clinical applications.


In first-line systemic therapy for unresectable recurrent and/or metastatic squamous cell carcinoma of the head and neck (R/M SCCHN), regimens are generally selected by time-to-relapse with 6 months cutoff after platinum (Pt)-containing definitive therapy, Pt-refractory or Pt-sensitive recurrence, but clinical characteristics between Pt-refractory and Pt-sensitive recurrence of R/M SCCHN has not been fully investigated. This study aimed to evaluate pattern of recurrence and efficacy for salvage treatment for recurrence after Pt-containing definitive therapy for R/M SCCHN in a real-world setting.We retrospectively reviewed 150 patients treated with Pt-containing definitive therapy and analyzed the pattern of recurrence and efficacy of salvage therapy for 63 patients with R/M SCCHN.p = 0.028) and in prognosis after the detection of recurrence for the overall population (p = 0.021), and for salvage treatment group (p = 0.023), and systemic therapy group (p = 0.003) between Pt-refractory and Pt-sensitive groups.Pt-refractory recurrence, Pt-sensitive recurrence, second primary cancer (SPC), and no relapse occurred in 23.3%, 18.7%, 14.7%, and 43.3% of patients, respectively. In the cases with distant metastatic recurrence, symptomatic recurrence was significantly more common in the Pt-refractory recurrence, while asymptomatic recurrence was significantly more common in the Pt-sensitive recurrence. The timing of detection of SPC was after 2 years in 59.0% of cases after the completion of definitive therapy and 63.6% of SPC were asymptomatic. There was a significant difference in ΔNLR2 (NLR after definitive therapy minus NLR at detection recurrence; Our analysis revealed the recurrence pattern after Pt-containing definitive therapy and showed the validity of dividing patients into Pt-refractory and Pt-sensitive recurrence with different prognosis in salvage therapy, especially systemic therapy. Patients with local advanced squamous cell carcinoma of the head and neck (L/A SCCHN) are treated with definitive therapy by surgery or radiotherapy (RT). Additionally, platinum (Pt) agents are key drugs in systemic therapy and are used in induction chemotherapy (ICT), chemoradiotherapy (CRT), or postoperative CRT (POCRT). However, even with these multimodality treatments, more than 50% of patients receiving definitive treatment develop locoregional and/or distant relapse within the first 2 years of initial treatment, and have a poor prognosis .The treatment strategy for recurrent or metastatic (R/M) SCCHN is dictated by the availability of locoregional treatment or sensitivity to Pt agents defined by the time-to-relapse in case of unresectable disease. Currently, two programed cell death-1 (PD-1) inhibitors, pembrolizumab (Pembro) and nivolumab (NIVO), were used as first-line systemic therapy worldwide for patients who are not amenable to local treatment. Pembro is used as a first-line chemotherapy for the Pt-sensitive recurrence or untreated patients without feasibility of local treatment , while nHere, we retrospectively evaluated the pattern of recurrence after Pt-containing definitive therapy and investigated the efficacy of salvage treatment for recurrence in patients with R/M SCCHN.We retrospectively reviewed the medical records of patients treated with definitive therapy between January 2010 and December 2019 at the University of Occupational and Environmental Health, Japan. In the present study, the inclusion criteria were as follows: (a) primary tumor site located in the head and neck; (b) histologically proven SCC; (c) stage III or IV according to the American Joint Committee on Cancer Staging System (Seventh edition); (d) treated with Pt-containing definitive therapy by CRT or surgery followed by POCRT; (e) diagnosis of R/M SCCHN. The exclusion criteria were as follows: (a) primary tumor site located in nasopharynx; (b) p16 positive oropharyngeal cancer.2 until August 2015, and weekly cisplatin 40 mg/m2 or triweekly cisplatin 80–100 mg/m2 after September 2015. A Pt agent was administered to patients with normal renal function above Cockcroft-Gault 60. The definitive radiotherapy dose was 70–72 Gy and the adjuvant radiotherapy dose was 60–66 Gy. Nutritional support was provided using prophylactic percutaneous endoscopic gastrostomy (PEG) before definitive radiotherapy. In cases without prophylactic PEG, nasogastric tube was used when patients had inadequate oral intake. All patients were supported with nutritional supplements. After the Pt-containing definitive therapy, patients were followed with a medical examination every 1–2 months and imaging examination by computed tomography (CT) including the neck and chest every 3–4 months for the first 2 years and followed with imaging examination by CT every 6 months from the third to the fifth years.Pt-containing definitive therapy was performed by surgery followed by POCRT or CRT. In cases, in which ICT with TPF (docetaxel/cisplatin/5-FU) was performed, Pt-containing definitive therapy was subsequently performed. The Pt agent in combination with RT was used daily carboplatin 25 mg/mWe collected the patients’ characteristics at definitive therapy and the detection of recurrence including age, sex, Eastern cooperative Oncology Group (ECOG) performance status (PS), primary tumor site, T and N status, disease stage, definitive therapy for primary tumor, radiotherapy, platinum agent used in definitive therapy, induction chemotherapy, nutrition support, body weight (BW), and neutrophil-to-lymphocyte ratio (NLR), symptoms at the time of detection of recurrence, detection modalities, and extent of recurrent disease.BW measurement and peripheral blood test were performed before definitive therapy, after definitive treatment, and at the time of detection of recurrence. NLR was calculated as the total neutrophil count divided by the total lymphocyte count. BW and NLR were defined as pre-BW and pre-NLR before definitive therapy, post-BW and post-NLR after definitive therapy, and rec-BW and rec-NLR at the detection recurrence, respectively. ΔBW1 and 2 were defined as pre-BW minus post-BW and post-BW minus rec-BW, respectively. ΔBW1% and 2% were calculated as a percentage of pre-BW and post-BW, respectively. ΔNLR1 and 2 were defined as pre-NLR minus post-NLR and post-NLR minus rec-NLR, respectively.In the salvage treatment, surgery was the first choice if resectable. In the case of unresectable disease, RT was selected if the field was not previously irradiated, and systemic therapy was performed if local treatment was difficult. Systemic therapy includes conventional cytotoxic chemotherapy, molecular targeted agents (cetuximab), and immune checkpoint inhibitors (ICIs) such as nivolumab or pembrolizumab. Nivolumab and pembrolizumab have been used in patients since March 2017 and December 2019, respectively. Best supportive care (BSC) alone was performed when it was difficult to treat with these salvage therapies. Their median follow-up period was 470 days .χ2-test, independent two-sample t-tests, or one-way analysis of variance were used to evaluate differences between two or three independent groups. The overall survival (OS) rate was estimated and plotted using the Kaplan−Meier method and compared using the stratified log-rank test. The data were censored on December 31, 2021. OS was calculated from the date of the start of salvage therapy to the date of death or the last confirmed date of survival. Risk factors of recurrence and of survival for salvage therapy were evaluated by estimating hazard ratios (HRs) and 95% confidence interval (CI) using univariate and multivariate COX proportional hazards models. SPSS version 27 software  was used for all statistical analyses. Statistical significance was defined as a p value <0.05.No statistical sample size calculations were conducted. Fischer’s exact test, the Of the 150 patients with L/A-SCCHN treated with Pt-containing definitive therapy, 65 patients (43.3%) who were free of recurrence, and 22 patients (14.7%) with a second primary cancer (SPC), and 63 patients (37.2%) with locoregional and/or distant metastasis as shown in p = 0.028) between the Pt-refractory and Pt-sensitive cohorts. BW decreased and NLR increased after the completion of definitive treatment. The results indicate the following: at the detection of recurrence, BW tended to decrease slightly in both groups, while NLR decreased in the Pt-sensitive recurrence and remained unchanged in the Pt-refractory recurrence. Clinical indicators of recurrence were analyzed using univariate analysis, but no significant factors were found in Patients’ characteristics at the detection of R/M SCCHN and SPC Table 2,p = 0.024), extent of recurrent disease , salvage treatment , and salvage surgery  were associated with a low risk of death. In multivariate analysis, time-to-relapse  and salvage treatment  were associated with a low risk of death.The univariate and multivariate analyses of OS after the detection of recurrence were presented in p = 0.021), and for salvage treatment group (p = 0.023), and systemic therapy group (p = 0.003). There were no significant differences between the Pt-refractory and Pt-sensitive recurrence for salvage surgery, RT, and BSC alone in In the Pt-refractory recurrence, first-line systemic therapy was performed by NIVO in 4 (44.4%) of 9 cases, but second-line systemic therapy was performed in 1 case only  had locoregional recurrence and/or distant metastasis, and the median time of recurrence was 166 days 95% CI: 117–215). The long-term follow-up data showed that locoregional recurrences developed in 16–25%  of patie–215. TheThis study showed an important finding of the frequency of asymptomatic recurrence according to pattern of recurrence. Asymptomatic recurrence occurred in 37.9% of locoregional recurrence only, 41.2% of distant metastasis, and 63.6% of SPC. Furthermore, asymptomatic distant metastasis after 6 months after definitive therapy occurred significantly more frequently and might require attention. Moreover, it is important to note that 63.6% of SPCs were asymptomatic recurrences and were found after 2 years in 59.0% of cases from the completion of definitive therapy. These suggest the effectiveness of planned imaging examination for follow-up after 6 months from the completion of definitive therapy to detect asymptomatic recurrence; however, the optimal frequency and modality of routine post-treatment imaging in asymptomatic patients is controversial . In thisIn the present study, ΔNLR2 was significantly different between the two cohorts and might be a useful indicator in detecting Pt-refractory recurrence. ΔNLR2 is post-NLR minus rec-NLR and was significantly lower in the Pt-refractory cohort. To the best of our knowledge, no study has examined the use of ΔNLR as a marker of recurrence of SCCHN. In the present study, there was no significant difference in rec-NLR between the two cohorts, but there was a higher NLR in the Pt-refractory cohort compared to the Pt-sensitive cohort. Elevated NLR has been reported to be associated with poor prognosis independent of tumor or disease stage in various cancer types, including HNC , 20. NLRΔBW1 (BW1%) was reduced by 4.8 kg (7.5%) in the no relapse group, and 5.1 kg (8.4%) in R/M SCCHN group, and 4.7 kg (8.3%) in SPC group, but the difference was not significant. In addition, ΔBW2 (BW2%) was further decreased by 1.4 kg (2.5%) in the Pt-sensitive recurrence and 1.6 kg (3.1%) in the Pt-refractory recurrence, but the difference was not significant. In both cohorts that had relapse, it might be important to note that BW was consistently decreasing throughout the treatment and follow-up periods. It has been reported that weight loss during treatment is associated with poor prognosis , 22. Perp = 0.023). This indicates that Pt-refractory patients have a poor prognosis even with salvage treatment. Furthermore, there was no significant difference in OS by salvage treatment between the Pt-refractory and Pt-sensitive recurrence, except systemic therapy. We believe that this result support the validity of dividing clinical trials in systemic therapy by time-to-relapse with 6 months cutoff. In systemic therapy of the Pt-sensitive recurrence, ICI was used in 66.7% of the cases, and the median OS was not reached because of the presence of long-term survivors by sequential treatment. All of these patients were selected for systemic therapy in April 2017, when ICIs first became covered by insurance in Japan, and no patients had received systemic therapy prior to that date. This suggests the usefulness of systemic therapy as an option when local treatment is not feasible now that ICI is available. However, Pt-refractory recurrence had a poor prognosis despite the use of NIVO in 44.4% of cases, indicating that patients with Pt-refractory relapse are a treatment-resistant population. Although there were no significant differences, the prognosis for the Pt-sensitive recurrence was good for surgery and radiotherapy compared to the Pt-refractory. The better response to salvage therapy might indicate a better prognosis for Pt-sensitive recurrence compared to the Pt-refractory recurrence. The result suggests that the two populations divided by time-to-relapse differ in their sensitivity to systemic therapy, requiring further exploratory studies.In R/M SCCHN, there is a history of clinical trial for systemic chemotherapy in which patients were divided into the Pt-refractory and sensitive groups , 12, 13 BSC alone had a worse prognosis compared to salvage treatment, and there was no significant difference between the Pt-refractory and Pt-sensitive recurrence. Indeed, these survival times for BSC alone were roughly equivalent to the 3.82 months reported for natural history of untreated head and neck cancer . Multivan = 150). However, to the best of our knowledge, this study is the first to investigate R/M SCCHN divided into Pt-refractory and Pt-sensitive recurrence after Pt-containing definitive therapy. The present results should therefore be considered to provide valuable information for selecting the treatment of R/M SCCHN patients. Second, salvage treatment modalities differ according to historical background, and the short duration of ICI resulted in a different prognosis between the two cohorts. Third, only SCCHN patients with recurrence were included with respect to their course after the completion of Pt-containing definitive therapy in this study. Therefore, although the usefulness of BW and NLR as markers of recurrence of SCCHN is unclear, it is useful to clarify the difference between the Pt-sensitive and Pt-refractory recurrence. Fourth, NLR and BW at 6 months after definitive treatment in the Pt-sensitive cohort were not measured, and differences in ΔNLR between the Pt-refractory and Pt-sensitive cohorts at 6 months after definitive therapy were unknown. However, we believe that the characteristics of Pt-refractory recurrence, in which the NLR that increased with definitive therapy did not decrease, might be clinically useful for the monitoring of recurrence. Dynamic changes in NLR during follow-up require further investigation. Fifth, we used time-to-relapse by the empirical “6 months” cutoff. In systemic therapy, regimens are commonly selected according to the Pt-refractory or Pt-sensitive recurrence as defined by time-to-relapse regimens. Although the biological characteristics of Pt-refractory and Pt-sensitive recurrence have not been clarified, the prognostic difference between the two groups has been revealed, and we consider that this is one of the useful results of this study. However, further investigation of biomarkers of Pt-refractory and Pt-sensitive recurrence is warranted.Several limitations of this study warrant mention. First, the study was conducted using a retrospective design at a single institution and with a small sample size (Our analysis revealed the pattern of recurrence after Pt-containing definitive therapy for L/A SCCHN and the frequency of Pt-refractory and Pt-sensitive recurrence. The clinical differences between Pt-refractory and Pt-sensitive recurrence were that ΔNLR2 was lower in the patients with Pt-refractory recurrence, while prognosis was better in those with Pt-sensitive recurrence in salvage treatment and systemic therapy groups. After Pt-containing definitive therapy, it is especially useful in systemic therapy to detect the recurrence by dividing into the Pt-refractory and Pt-sensitive recurrence, which have different prognosis.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.Ethical review and approval was not required for the study on human participants in accordance with the local legislation and institutional requirements. The patients/participants provided their written informed consent to participate in this study.TW designed the study and wrote the initial draft of the manuscript. HS contributed to analysis and interpretation of data and assisted in the preparation of the manuscript. All other authors have contributed to data collection and interpretation, and critically reviewed the manuscript. All authors approved the final version of the manuscript, and agree to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


For the first time, a novel aptamer was designed and utilized for the selective detection of rivaroxaban (RIV) using the integration of bioinformatics with biosensing technology. The selected aptamer with the sequence 5′-TAG GGA AGA GAA GGA CAT ATG ATG ACT CAC AAC TGG ACG AAC GTA CTT ATC CCC CCC AAT CAC TAG TGA ATT-3′ displayed a high binding affinity to RIV and had an efficient ability to discriminate RIV from similar molecular structures. A novel label-free electrochemical aptasensor was designed and fabricated through the conjugation of a thiolated aptamer with Au nanoparticles (Au-NPs). Then, the aptasensor was successfully applied for the quantitative determination of RIV in human plasma and exhaled breath condensate (EBC) samples with limits of detection (LODs) of 14.08 and 6.03 nM, respectively. These valuable results provide ample evidence of the green electrogeneration of AuNPs on the surface of electrodes and their interaction with loaded aptamers (based on Au-S binding) towards the sensitive and selective monitoring of RIV in human plasma and EBC samples. This bio-assay is an alternative approach for the clinical analysis of RIV and has improved specificity and affinity. As far as we know, this is the first time that an electrochemical aptasensor has been verified for the recognition of RIV and that allows for the easy, fast, and precise screening of RIV in biological samples. In clinical practice, atrial fibrillation (AF) is the most prevalent persistent heart arrhythmia disease. Stroke, thromboembolism, heart failure, and poor quality of life are all significant causes of death in people with AF and its possible complications ,2,3. TheRIV, chemically identified as 5-chloro-N-({(5S)–2-oxo-3-[4-(3-oxomorpholin-4-yl) phenyl] 1, 3-oxazolidin-5-yl methyl) thiophene-2-carboxamide ) is an oIn the pH range of 1 to 9, RIV is virtually insoluble in water and aqueous solutions and is prescribed in fixed doses  to all patients . Rarely,RIV is an oral, selective, and direct factor Xa (FXa) inhibitor, and it inhibits both free and bound FXa in the prothrombinase complex, with a rapid onset of action . It has Although RIV has predictable pharmacokinetics and pharmacodynamics, there is still a strong tendency to measure and monitor RIV in some clinical circumstances . TheAlthough the above-mentioned methods provide simultaneous and accurate analytical results with low limits of detection (LODs) for RIV recognition, they do demand sophisticated instruments, tedious sample pre-treatment procedures, long processing times, high analysis costs foreach sample, complicated manipulation, and expert technical skills that restrict their application for the rapid monitoring of RIV ,46. As aTo achieve this goal, biosensors based on aptamers are presented as a promising platform that can identify different targets by producing a measurable physical signal. Aptamers are short and single-stranded DNA or RNA oligonucleotides that can selectively bind to a specific target, including proteins, peptides, carbohydrates, small molecules, toxins, and even live cells.d) (from picomolar to nanomolar), aptamers have become ultra-selective recognition elements compared to other existing molecules for biochemical assay. Aptamers have been widely used for various sensor-based approaches, such as in electrochemical, colorimetry, fluorescent surface, and plasma resonance-based biosensors ·3H2O), potassium ferricyanide (K3[Fe(CN)6]).3H2O, anhydrous acetic acid, EDC/NHS (1-ethyl-3-(3-dimethylaminopropyl) the carbodiimide/N-hydroxysuccinimide), Tris-HCl (tris(hydroxymethyl)aminomethane hydrochloride), cetyltrimethylammonium bromide (CTAB), MCH (mercaptohexanol), H2SO4 (sulfuric acid), ascorbic acid, arginine, glucose, L-cysteine, glycine, tyrosine, uric acid, glutamic acid, magnesium, L-proline, hydrochlorothiazide, metoprolol, nitroglycerin, aspirin, and losartan and KCl (potassium chloride) were obtained from Merck . An indium tin oxide–poly polyethylene terephthalate (ITO-PET) electrode . PCR master mix (2×) and the DNA ladder were purchased from the company Amplicon. Deionized and pyrogen-free water was obtained from a Milli-Q water purification system . Nuclease-free water was used to make all of the aqueous solutions. All chemicals were at the maximum purity obtainable unless otherwise noted.Epoxy-activated Sepharose 6B and the columns were obtained from Thermo Scientific and were purchased from GE Healthcare . The QIAEX II Gel extraction kit and QIAprep spin miniprep kit were purchased from QIAGEN Inc. . Low-melting-point agarose was obtained from Fermentas , and the pGEMDeionized water and human plasma samples were prepared by the Ghazi Pharmaceutical Company  and the Blood Transfusion Research Center , respectively. Sample donors signed a written consent form approved by the ethics committee of the Tabriz University of Medical Science (IR.TBZMED.VCR.REC.1397.317).−1 in order to investigate the nature of the chemical bonds. (ATR-FTIR) Analysis: Attenuated total reflection Fourier transform infrared (Bruker Vertex 70 ATR Model) was used to monitor the functional groups on modified ITO-PET electrode surfaces using an ATR apparatus. The measurements were recorded in the range of 400–4000 cmThe morphology of the bulk materials and modified ITO-PET electrode surfaces were characterized by a high-resolution field emission scanning electron microscope  with an operating voltage of 3 kV, and the chemical compositions of the electrodes were analyzed by energy dispersive spectroscopy (EDX) coupled with FE-SEM equipment.−1, with a resolution of 256 pixels per line. Electrochemical measurements were carried out in an ordinary three-electrode cell  powered by an electrochemical system comprising a Palm Sense 4c system with PS4.F1.05 . The three-electrode system consisted of Ag/AgCl-saturated KCl  as a reference electrode, a platinum wire, and a modified ITO-PET electrode with Au nanoparticles (2 mm × 20 mm), which were utilized as the counter and working electrodes, respectively. Chronoamperometry (ChA) and square-wave voltammetry (SWV) analyses were performed in the presence of 0.01 mM of K3 [Fe(CN)6]/K4 [Fe(CN)6] (1:1) mixture and 0.1 M KCl. For the ChA measurements, tequilibrium time = 2, interval time = 0.1 s, run time = 200 s, and EDC =0 V were employed. In SWV technique investigations, the scan rate was adjusted for 0.05 V s−1, the modulation amplitude was 0.25 mV, the step potential was 0.01 mV, and the potential range was −1 V to 1 V. All measurements were the average of at least three replicates.AFM Analysis: Atomic force microscopic analyses were recorded using Nanosurf  in tapping mode to confirm the surface immobilization of the aptamers on the substrates. The scan speed was 2 μm.sThe quantity and quality of the isolated DNA were analyzed using a Nanodrop 1000 . Chromatographic analysis was performed using Alliance separations module 2795 , which consisted of a quaternary solvent delivery system with a vacuum degasser pump with a C18 column (60 × 4.6 mm × 5 microns) from the Agilent Company. The confirmatory analysis of the blank sample was performed on the same apparatus coupled with a Quattro Micro quadrupole mass spectrometer  equipped with an electrospray source (Z-spray). During the analysis of the samples, the flow rate of the mobile phase (methanol: water at a ratio of 65:35) was adjusted to 0.6 mL/min. SPR measurements were conducted using a SRPNavi 210A device  with gold chips .Synthesis of the acidic derivative of RIV was performed based on the previously reported method . For thi1H NMR : δ (ppm): δ = 3.43 , 3.64 , 3.76 , 3.90 , 4.2 , 4.12 , 4.20 , 4.86 , 7.21 , 7.38 , 7.58 , 7.77 , and 9.12 .After the completion of the reaction, the suspension was kept at room temperature for 15 h. The resulting crystals were filtered and washed by suction with 0.08 mL of acetic acid. For better filtration, crystals were suspended twice in 0.3 mL of isopropanol and were washed again with a suction filter and again with 0.4 mL of isopropanol. The remaining crystals remained at 35 °C for 15 h at a pressure of less than 80 mbar to obtain a pure product as a light brown solid. Yield: 0.93 g (85%). The amination of epoxy-activated Sepharose 6B was performed in the presence of hexamethylene diamine according to the method of Teng et al. . Under vFirst, deionized water was used to wash the replaced Sepharose. A mixture of RIV acidic derivative (8.6 mmol) and carbonyldi-imidazole CDI (0.8 mmol) in 2 mL of DMF were stirred for 1 h at room temperature with a magnetic stirrer under argon gas . Then, t−1 (128 scans) over the 400–4000 cm−1 range, with and without baseline corrections. Scanning electron microscopy (SEM) images of both the derivatized RIV gel and epoxy-activated Sepharose were acquired in a Hitachi S-2700 with a UHV Dewar detector (Rontec EDX) . The samples were magnified 150, 450, and 4000×. Before all of the analyses, the samples were well-dried at 50 °C in the presence of phosphorus pentoxide.The Fourier transform infrared spectra (FT-IR) of the RIV derivatized gel and of the epoxy-activated Sepharose (with no RIV) were acquired on a Thermo Scientific Nicolet iS10 FT-IR spectrometer . Spectra were recorded at 4 cmThe random DNA library contained central randomized sequences of 20, 30, or 40 nucleotides (N20 or N30 or N40) flanked by a 23-nucleotide constant region: 5′ -TAG GGA AGA GAA GGA CAT ATG AT(N20 or N30 or N40) TTG ACT AGT ACA TGA CCA CTT GA-3′. Strand primer up (5′-TAG GGA AGA GAA GGA CAT ATG AT-3′) and a3′ (-) strand primer down (5′-TCA AGT GGT CAT GTA CTA GTC AA-3′) were prepared using Micro synth AG . The library was used as a template DNA for the synthesis of single-stranded DNA (ssDNA) in asymmetric PCR.2, pH = 7.4). Next, 50 μL (50 pmol/μL) of the synthetic ssDNA library was dissolved in 200 μL of binding buffer and was slowly denatured at 95 °C for 10 min before being quickly cooled on ice for 10 min. Then, the appropriate quantity of RIV-immobilized beads (2 mL) was added to the above hybrid library solution and incubated with gentle agitation (1 h at room temperature) for immobilization. The medium was washed with wash buffer  to remove the unspecific and unbound DNA. Additionally, the bound ssDNAs were then eluted in binding buffer with four column volumes with 5 to 0.5 mM of RIV. The recovered ssDNA was amplified by asymmetric PCR. The 25 μL PCR amplification was made of 10 pg ssDNA template, forward primer (0.4 μM), reverse primer (0.004 μM), and 10 µL PCR master mix (2×). The special aptamers for RIV were selected and extracted at the end of the SELEX cycles by affinity chromatography on a RIV–Sepharose column ). BeforeE. coli bacteria. Plasmid extraction was conducted according to the qiagene miniprep kit protocol. After controlling for positive clones by PCR, the samples were sent to be sequenced by macrogene Korea /K4[Fe(CN)6] and KCl (0.01 M) (1:1) as a redox probe. In the CV experiments, the working potential range was between −500 and 500 mV at a scan rate of 100 mVs−1.During the last step of fabrication, after being properly washed with ultrapure water, the acquired ITO-PET-OH-AuNPs-aptamer-MCH-functionalized ITO substrate was ready for RIV measurement. Finally, the prepared bioelectrode was used for RIV detection. Electrode preparation steps, optimization studies, and the analytical behavior of developed aptasensor were studied by following the electrochemical CV technique. All of the electrochemical experiments were performed in a solution containing K3[Fe(CN)6] and K4[Fe(CN)6] showed almost completely irreversible behavior on the (a) bare ITO-PET, (b) ITO-PET-OH, (c) ITO-PET-OH-AuNPs, (d) ITO-PET-OH-Au NPs-aptamer, and (e) ITO-PET-OH-Au NPs-aptamer /RIV (1 mM) electrodes when a potential sweep rate of 100 mVs−1 had been employed. According to the obtained results, the oxidation peak of the EC probe on the surface of ITO-PET has an intensity of 1198.74 µA, showing a higher oxidation peak current than the other electrodes. When the bare electrode was modified by OH, the peak current dropped greatly (559.3 µA) and validated the surface-blocking redox process’ hindrance behavior [Fe (III)/Fe (IV)]. After the gold nanoparticles were electrogenerated on the surface of ITO-PET-OH, the peak potential shifted to 0.549 V, and the peak current increased to 814.89 µA. This result shows that the electrodeposition of the Au NPs on the surface of ITO PET-OH and the excellent features of Au NPs, such as electrical conductivity and a large specific surface area, allowed more (Fe(CN)6)3−/4− to reach its surface and electron transfer to occur at a higher rate. Certainly, the Au nanoparticles stabilized on the ITO-PET-OH film reveal appropriate catalytic characteristics as well as increased the rate of electron transfer and demonstrated appropriate electroactivity. Therefore, the gold nanoparticles are promising candidates for forming an aptamer-active substrate.The electrochemical behavior of the aptasensor preparation steps was clearly investigated via the CV technique in 0.01 M of ferrocyanide/ferricyanide containing KCl solution as a mediator, as it provides a simple and effective method for evaluating electron transport between the solution and the electrode surface. As shown in In contrast, when the aptamers were assembled on the ITO PET-OH-modified electrode via the AuNPs reaction, the peak currents of ITO PET-OH-AuNPs-aptamer obviously decreased, which led to a decrease in the peak height (from 0.5 to 0.6 V and from 814.89 to 53.16 μA in DPV the plots). This result indicates that as a biological element, aptamers (bioreceptors) were successfully immobilized on ITO-PET-OH-AuNPs. Because aptamers are macromolecule and occupy a large portion of the electrode surface, it reduces the electron transfer of ferro/ferry (supporting electrolytes). In reality, aptamers function as an insulator, slowing down electron transmission.6]3−/4− at the electrode surface, and the interfacial charge transfer was increased. This means that the aptamer has completely covered the electrode’s surface and that the signal is set as Signal ON.According to the findings, in the presence of RIV targets, it was observed that the anodic peak current increased to about 601.36 µA. This indicates that in the presence of RIV, as a result of the formation of a RIV/aptamer complex on the surface of the electrode, the aptamer folds due to RIV binding; therefore, the presence of a higher negative charge (both of the aptamer phosphate group and the AuNPs) resulted in a larger repulsion of 3−/4− solution under optimized conditions in a standard sample. The relative electrochemical signal increased gradually upon the enhancement of the RIV concentration and an excellent linear relationship between the current response signal and the RIV concentration was achieved. The calibration curve was linear over a concentration range of 10–500 nM, with a regression equation of (I/μA) = −0.4058 C (RIV) + 276.88 and R2 = 0.9917. The signal-to-noise ratios (S/N) of 3 and 10 were used to calculate the LOD and LOQ, respectively. The LOD and LOQ values were found to be 6.6 and 21.9 nM, respectively ectively A,B.In The majority of the reported HPLC techniques have various flaws, including low sensitivity, complicated mobile phases, and time-consuming extraction procedures with restricted applicability, as illustrated in Regarding Prior research indicates that the sensitivity of HPLC-UV and LC-MS/MS in the examination of RIV is nearly equal  despite As we know, these methods need to be run by an expert operator. Additionally, in HPLC-based analytical techniques, samples need to go through pretreatment steps. As such, samples should be prepared for inquiry according to the appropriate the pretreatment steps, including extraction, concentration, dilution, clean-up, and derivatization. For this purpose, solid-phase extraction (SPE), protein precipitation (PP), and liquid–liquid extraction (LLE) are often considered ,84.® Turbo Flow® technology used in this assay was unusual and is not found in most laboratories 3−/4−/KCl (0.01 M)] as the supporting electrolyte. For this purpose, 5 µL of unprocessed human plasma and EBC samples were mixed with various amounts of RIV and were incubated on the surface of the ITO-PET-OH-AuNPs-aptamer.To assess the ability of the proposed aptasensor to detect RIV in biological samples, the performance of the sensor to measure RIV in human plasma and EBC samples was investigated using the ChAs technique and 3−/4−/KCl (0.01 M)]. As illustrated in Undoubtedly, one of the most striking properties of a biosensor is the ability to measure the targets in a sample containing similar species as interfering compounds. Multiple potentially interfering species in real samples that may interfere in the determination of RIV, such as ascorbic acid (AA), arginine (Arg), glucose (GlC), L-cysteine (L-Cys), glycine (Gly), tyrosine (Tyr), uric acid (UA), glutamic acid (Glu), magnesium (Mg), and L-proline (Pro), were prepared in standard solution to evaluate the aptasensor’s selectivity in the supporting electrolyte ). To ach2+, and glucose have the lowest current intensity among the selected species, this indicates that they have no interaction with the desired aptamer, which may be due to their small size. So, the coexistence of these species certainly does not cause interference with RIV detection.It is important to emphasize that the constructed aptasensor did not show significant responses to other tested compounds. Whereas L-Gly, MgTherefore, adding interfering substances resulted in no discernible increase in the peak current, and the interference was nearly negligible, indicating that the fabricated aptasensor exhibits remarkable specificity for the recognition of RIV.t-test was performed to check the significance of the data using SPSS Statistics version 20.0 software. With all of the tested drugs, apart from aspirin (p = 0.042), no statistically significant differences (p < 0.05) were obtained between the responses  that could be co-administrated with RIV. The drugs were prepared in the standard solution and were evaluated in the supporting electrolyte. The anti-interference performance of the sensor was evaluated by measuring the RIV peak current value in the presence of the selected drugs and comparing it to that of the RIV response in the absence of the drugs. A paired esponses . This coStability is the ability to efficiently reuse a sensor and is a prominent factor in the evaluation of engineered aptasensor performance. Indeed, the aim of the stability testing method is to determine whether or not the analyte is stable with changes in the environment, such as changes in the matrix and storage conditions. For this purpose, the aptasensor’s stability in relation to the number of cycles was assessed by monitoring the drop in the peak current intensity during repeated CV using an ITO PET-OH-AuNPs electrode.6]3−/4−/KCl (0.01 M)] solution. The results indicated that there was little substantial difference between the first cycle, the fifth cycle, and the tenth cycle because the peak currents of the aptasensor retained high stability when the number of cycles increased to 10 and then decreased very smoothly. However, with a rise in the number of cycles to 50, the peak current decreased intensively. This indicates that after 50 cycles, the electrode surface is practically inactive and confirms the instability of the interface. According to these findings, the related electrode has excellent performance and repeatability for up to ten cycles, and the engineered substrate can be used for up to ten cycles.6]3−/4−/KCl (0.01 M)] solution. According to For practical applications, the stability of the engineered aptasensor is crucial. The intra-day stability of the electrode was studied using one electrode by putting it in the fridge for three consecutive days and recording the CVs. The electrochemical behavior of the developed aptasensor was studied by recording the CVs in the supporting electrolyte [Fe(CN)Using an affinity chromatography-based SELEX strategy, we have identified, developed, and characterized a novel aptamer that can bind to RIV with a high level of affinity and specificity for the first time. After that, the RIV-specific aptamer was used to develop and fabricate a label-free electrochemical nano-aptasensor for RIV detection in human plasma and EBC samples with LOD values of 14.08 and 6.03 nM, respectively, and a wide linear range of 10 nM to 500 nM. RIV determination was carried out under the optimized conditions. Therefore, the proposed aptasensor is able to quantify the RIV in human plasma and EBC as biological samples with a low LOD, a wide linear detection range, good selectivity, and high stability. Taking into account the therapeutic levels of RIV, which lie between 27.5 and 575.5 nM , this se